Cofactors may be important determinants, acting as nucleation points limiting the conformational search, for the folding rates of cofactor-binding proteins. To date, folding kinetics of proteins with b-sheet structure has not been as thoroughly investigated as for helical proteins. This research program aims towards probing the role of two inorganic and one organic cofactor in the folding of three proteins with mostly b-sheet structure. The targeted proteins are azurin, a b-barrel protein with a copper-ion cofactor, flavodoxin, a protein with an a/b doubly-wound topology coordinating an organic flavin mononucleotide (FMN), and cytochrome f, a b-sheet protein covalently linked to a heme. Equilibrium biophysical characterization (circular dichroism, fluorescence, absorption, EXAFS, NMR, and various biochemical methods) will aid in revealing the effect of each cofactor on its corresponding protein stability and unfolded polypeptide structure. A recent technique in which folding is initiated by photochemical electron-transfer will be used to probe rapid events during formation of the native-states of the proteins. To allow wide denaturant- and time- ranges to be investigated (and to study the apo proteins), time-resolved experiments will also be performed using stopped-flow mixing. The specific aims are to: 1. Characterize cofactor- coordination and residual structures created by the cofactors in the unfolded states, 2. Investigate how copper, FMN and heme (bound to the unfolded polypeptides) affect the polypeptide folding kinetics and, finally, 3. Probe early events (starting on the us time scale) during the formation -of native azurin, flavodoxin and cytochrome.